Versatile continuous manufacturing platform for cell-free chemical production

ABSTRACT

The present invention features a versatile continuous manufacturing platform for cell-free chemical production.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a non-provisional and claims benefit of U.S.Provisional Application No. 63/127,758 filed Dec. 18, 2020, and U.S.Provisional Application No. 63/127,836 filed Dec. 18, 2020, thespecifications of which are incorporated herein in their entirety byreference

FIELD OF THE INVENTION

The present invention relates to devices and methods for the productionof chemicals in a cell-free continuous manufacturing platform.

BACKGROUND OF THE INVENTION

A natural product is defined as being a molecule found in Nature createdfrom a natural process. These broad classes of molecules find use astherapeutics, agrochemicals, or industrial starting materials. Thenatural processes that form these materials are typically multi-stepenzyme pathways. Such enzyme pathways convert simple starting materialssuch as glycerol and glucose into complex materials through multi-stepenzyme reactions. Currently, the majority of natural products arecultivated and extracted from plants, synthesized via complex chemicalsynthesis, or biomanufactured through cell-based factories also known asbiofoundries. The present invention details the workings of a scalablecontinuous system to house immobilized enzymes that mimic how Naturecreates diverse ranges of natural products.

Manufacturing natural products via cultivation, chemical synthesis, orthe use of modified cells suffers from many problems that limitcommercial viability of bio-based specialty chemical industrialization.First, cultivation requires vast amounts of land/energy/water, and theplant is only capable of producing the high value material in lowamounts. Next, chemical synthesis requires extensive, elaborate,expensive, toxic, and inefficient multi-step chemical reactions toproduce natural products that often are too complex to make in thelaboratory. Finally, the cell suffers from product toxicity, carbon fluxredirection, diffusion problems through cell walls, and toxic byproductgeneration. To overcome these problems, the use of enzymes in thepresent system provides a viable alternative. As the same enzymes areused as the cell, but without the limitations of the cell, this researchhas been dubbed ‘cell-free’.

However, for cell-free manufacturing to be competitive new technologymust be developed to allow each enzyme to experience its optimalreaction conditions to drive higher titers.

BRIEF SUMMARY OF THE INVENTION

It is an objective of the present invention to provide devices andmethods that allow for the production of chemicals in a cell-freemanner, as specified in the independent claims. Embodiments of theinvention are given in the dependent claims. Embodiments of the presentinvention can be freely combined with each other if they are notmutually exclusive.

In a provisional patent application entitled “CELL-FREE PRODUCTION OFGERANYL PYROPHOSPHATE FROM GLYCEROL IN A CELL-FREE MANUFACTURINGSYSTEM”, the inventors demonstrated glycerol conversion to geranylpyrophosphate (GPP) in a batch reaction using immobilized andnon-immobilized enzymes in a prior provisional patent. Here, 12individual enzymes were placed into a reactor and mixed for five days toafford 49 mg/L of GPP (FIG. 1). However, using these differentimmobilized enzymes in a single batch operation confines the chemistryopportunity. Because only one reaction temperature, time, and pH couldbe accommodated in a single reactor, the reaction efficiency would behindered. Additionally, to draw on the benefits of continuousmanufacturing with this number of enzymes would require creating andoptimizing a system from first principles.

The individual requirements of the 12 immobilized enzymes involved inthe biochemical pathway to convert glycerol into geranyl pyrophosphatewere explored first (Table 1); each enzyme has a unique set ofconditions needed for optimal reactivity. Furthermore, deviation awayfrom optimal conditions would lead to loss of reactivity through proteinprecipitation as well as product and substrate degradation; factors thatwould leave this process unscalable and commercially unviable. Thus, asystem was needed that could provide these individual enzymerequirements, could overcome protein precipitation, could providemultiple temperatures, multiple pH's, multiple reaction times, anddifferent reaction solution oxygenation levels at any given time. Ineffect, it was impossible to satisfy all the individual enzymerequirements in the current manufacturing equipment of today. Toovercome this limitation, the 12 immobilized enzymes were placed intocontinuous manufacturing reactors that are strictly controlled by anarray of external sensors and algorithmic feedback loops to createoptimal reaction conditions and for these enzymes and others in thefuture.

TABLE 1 The optimal reaction conditions for each enzyme involved in thebiochemical pathways converting glycerol into GPP and then through toCBGA through use of a reporter enzyme system (NphB enzyme). In thesereactions, between 10-100 mg of enzyme- resin complex was used, and thereaction was monitored through use of high-performance liquidchromatography (HPLC) coupled with a refractive index detector (RID).Reaction Conditions Screened Temp Time Yield Enzyme Name (° C.) pH (h)(%) MBP-Aldo (Aldo) 37 9.0 21 98 Dihydroxy Acid Dehydratase 45 8.0 16 32(TvDHAD) Pyruvate Oxidase (PyOx) 37 6.5 16 96 Acetyl-phosphatetransferase 32 8.0 8 60 (PTA) Acetyl-CoA acetyltransferase 32 8.0 8 44(PhaA) HMG-CoA Synthase A110G 32 7.5 2 54 (HMGS) HMG-CoA Reductase(HMGR) 37 7.0 2 55 Melvonate Kinase (MVK) 37 8.0 — 87 PhosphomevalonateKinase 37 8.0 32 96 (PMVK) Diphosphomevalonate Kinase 37 8.0 16 94 (MDC)Isopentyl-PP Isomerase (IDI) 22 8.0 2 28 Farnesyl-PP synthase S82F 258.3 4 81 (FPPS) Prenyl transferase (NphB) 50 8.0 6 16

The inventors have created a versatile continuous manufacturing platformthat allows cell-free biomanufacturing to be scaled while providing thenecessary conditions for the enzyme reactions to work. This patentapplication describes the manufacturing system and its use in animportant biomanufacturing approach.

As described herein, a cell-free system and the key reactor drivers (seeTable 1) are used in a cell-free chemical reaction (i.e., without thecell being present). The required enzymes are first created in vivo(typically through protein overexpression), isolated via chromatography,and then added into a bioreactor with a low-cost substrate. The enzymestransform the low-cost substrate into product via the exact same waythat occurs in plants, animals, and bacteria but without the complexityof the organism. In this way, natural pathways can be harnessed tocreate natural molecules.

In some embodiments, the present invention features a cell-freecontinuous manufacturing platform for chemical production. In someembodiments, the platform comprises one or more individual reactors anda pumping system adapted to flow a solution through the one or moreindividual reactors. In some embodiments, each of the one or moreindividual reactors comprises a cylindrical tube comprising a first endand a second end. In some embodiments, both the first end of thecylindrical tube and the second end of the cylindrical tube comprisefittings (i.e., stainless steel fittings). In some embodiments, acylindrical tube interior of the individual reactor comprises a resinand an enzyme. In some embodiments, each of the one or more individualreactors has an input tubing connected at the first end of thecylindrical tube and an output tubing connected at the second end of thecylindrical tube to create a closed system. In some embodiments, thecylindrical tube interior of the individual reactor further comprisesone or more sensors.

One of the unique and inventive technical features of the presentinvention is the use of cell-free manufacturing. Without wishing tolimit the invention to any theory or mechanism, it is believed that thetechnical feature of the present invention advantageously provides forhigher reaction concentrations, no cell-wall to battle for product andsubstrate diffusion, no battling the cell for carbon flux and byproductformation, no cell death due to the formation of toxic compounds asthere is no cell, and cell-free offers a platform solution to create alarge number of compounds; cells have to be re-programmed and thisinvention simply allows a reactor column to be switched out for anotherone containing a different immobilized enzyme. None of the presentlyknown prior references or work has the unique inventive technicalfeature of the present invention.

Furthermore, the prior references teach away from the present invention.For example, the current use of immobilized enzymes typically use asingle reactor (batch or continuous) that only allows for one set ofreactor conditions (time, pH, temperature, etc.) The present inventionallows for the use of different reactor conditions between eachindividual reactor (or reactors in sequence) without intermediateisolation. Moreover, the addition of gases via a controlled moduleallows for enzymes requiring oxygen (or a lack of) to be used in acontinuous reactor, which previous devices have not been able to do.Additionally, the deoxygenation module allows oxygen “phobic” and oxygen“philic” enzymes to be used in sequence, again, without intermediatepurification.

Furthermore, the inventive technical features of the present inventioncontributed to a surprising result. For example, the device of thepresent invention can fit the same amount of enzymes in a 79 mLcontinuous reactor as compared to a 1,000 L traditional fermenter,allowing for a higher reaction concentration. When a 45 L reactor isachieved, this will be the equivalent of a 567,000 L traditionalfermenter.

Any feature or combination of features described herein are includedwithin the scope of the present invention provided that the featuresincluded in any such combination are not mutually inconsistent as willbe apparent from the context, this specification, and the knowledge ofone of ordinary skill in the art. Additional advantages and aspects ofthe present invention are apparent in the following detailed descriptionand claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

The features and advantages of the present invention will becomeapparent from a consideration of the following detailed descriptionpresented in connection with the accompanying drawings in which:

FIG. 1 shows the biochemical pathway to create GPP from glycerol thathas been previously shown. Here glycerol is transformed into GPP using12 individual enzymes, as described herein.

FIG. 2 shows the optimal reaction temperature for DHAD. Here, 45° C. isthe optimal temperature with significant drop off in yield observed whenmoving away from 45° C.

FIG. 3 shows a reactor contained within a housing with heating/coolingPeltier elements attached and supporting electronics circuits.

FIG. 4 shows the Grafana Data dashboard showing temperature andelectrical output data of four reactors over time along with eachreactor setpoint.

FIG. 5A shows the pressure over time (24 hrs) of the first reactor inthe system, one sample per second. Mean=0.092 PSI.

FIG. 5B shows the pressure over time (24hrs) as seen by the pHadjustment pump between the first two reactors in the system. Mean=0.789PSI.

FIG. 6 shows a screen capture of the graphical user interface (GUI).

FIG. 7A shows a standard voltage divider circuit. As R2 changes, V_(out)changes.

FIG. 7B shows the standard equation to calculate output voltage(V_(out)) based on input voltage (V_(in)) and resistor values (R1, R2)

FIG. 8 shows a simplified version of the Steinhart-Hart equation used toconvert thermistor resistance to a temperature value.

FIGS. 9A-9B show a Circuitry for Temperature Control, Standard (FIG. 9A)or if both heating and cooling is required without reorienting thePeltier elements (FIG. 9B).

FIG. 10A shows a thermal image of four reactors mounted and beingheated/cooled.

FIG. 10B shows a thermal image of a reactor being cooled to 12° C.

FIG. 11 shows a graph of dissolved oxygen removal in liquid water as afunction of time. The probe was placed in a reservoir of deionizedwater, then was placed into treated deoxygenated/degassed water and wascontinuously stirred. Dissolved oxygen went from 7.75 ppm to 4.07 ppm, areduction of almost 50%.

FIGS. 12A-12D show ¼″ and ¾″ Outer Diameter Reactor Housings—¼″ OuterDiameter Reactor Housing Tapped Side (FIG. 12A) or Screw Side (FIG. 12B)and a ¾″ Outer Diameter Reactor Housing—Tapped Side (FIG. 12C), or ScrewSide (FIG. 12D).

FIG. 13 shows a data flow from capture to display.

FIG. 14 shows a reaction set-up for the reaction in example 2.1.

FIG. 15 shows a reaction set-up for the reaction in example 2.2.

FIG. 16 shows a reaction set-up for the reaction in example 2.3.

FIG. 17 shows a reaction set-up for the reaction in example 2.4.

FIG. 18 shows a reaction set-up for the reaction in example 2.5.

FIG. 19 shows a reaction set-up for the reaction in example 2.6.

FIG. 20 shows a reaction set-up for the reaction in example 2.7.

FIG. 21 shows a reaction set-up for the reaction in example 2.8.

FIG. 22 shows a reaction set-up for the reaction in example 2.9.

FIG. 23 shows a reaction set-up for the reaction in example 2.10.

FIG. 24 shows a reaction set-up for the reaction in example 2.11.

FIG. 25A shows the reaction set-up for removal of oxygen from areaction. A pump pushes liquid through all four channels of adeoxygenation/degassing machine prior to entering a reactor.

FIG. 25B shows a diagram illustrating the addition of nitrogen gas priorto being pumped through the deoxygenation and/or degassing module and areactor.

FIG. 26 shows a reaction set-up for the reaction in example 2.13.

FIG. 27 shows a testing set-up for achieving equal throughput of twoparallel reactors connected to a single pump.

FIG. 28 shows commercial software to visualize the wavelength intensityacross the spectrum for a volume flowing through the flow cell that isconnected to the spectrometer.

FIG. 29 shows the output of software written to capture and/or displaythe absorbance data across the spectrum for a volume flowing through theflow cell that is connected to the spectrometer.

FIG. 30 shows several views of an individual reactor as describedherein.

FIG. 31A and 31B show 2D diagram of a cell-free manufacturing platformas described herein. FIG. 31A shows a cell-free manufacturing platformdescribed herein comprising a pH module. FIG. 31B shows a cell-freemanufacturing platform described herein comprising a pressure sensor anda gas addition module or degassing module.

FIG. 32 shows one embodiment of a cell-free manufacturing platform asdescribed herein. FIG. 32 shows a 3D cell-free manufacturing platformdiagram comprising 4 individual reactors (i.e., four reactions) with apH control module attached.

FIG. 33 shows a circuit schematic that can be used to heat/coolthermoelectric coolers or heat electric silicon heaters.

FIG. 34A and 34B show certain embodiments of the present invention asdescribed herein. FIG. 34A shows a 2D diagram of the individual reactorof the cell-free manufacturing platform as described herein and FIG. 34Bshows a 2D diagram of the cell-free manufacturing platform as describedherein.

DETAILED DESCRIPTION OF THE INVENTION

Before the present compounds, compositions, and/or methods are disclosedand described, it is to be understood that this invention is not limitedto specific synthetic methods or to specific compositions, as such may,of course, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only andis not intended to be limiting.

Additionally, although embodiments of the disclosure have been describedin detail, certain variations and modifications will be apparent tothose skilled in the art, including embodiments that do not provide allthe features and benefits described herein. It will be understood bythose skilled in the art that the present disclosure extends beyond thespecifically disclosed embodiments to other alternative or additionalembodiments and/or uses and obvious modifications and equivalentsthereof. Moreover, while a number of variations have been shown anddescribed in varying detail, other modifications, which are within thescope of the present disclosure, will be readily apparent to those ofskill in the art based upon this disclosure. It is also contemplatedthat various combinations or sub-combinations of the specific featuresand aspects of the embodiments may be made and still fall within thescope of the present disclosure. Accordingly, it should be understoodthat various features and aspects of the disclosed embodiments can becombined with or substituted for one another in order to form varyingmodes of the present disclosure. Thus, it is intended that the scope ofthe present disclosure herein disclosed should not be limited by theparticular disclosed embodiments described herein.

As used herein, the singular forms “a,” “an,” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including,”“includes,” “having,” “has,” “with,” or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising.”

As used herein, a “reactor” or an “individual reactor” may refer to acontinuous reactor containing an enzyme-resin complex. An individualreactor means one reactor with an entry and exit as defined by the fluidentering or leaving the reactor, respectfully. Additionally, two or moreindividual reactors may be linked together to form a reactor system.

As used herein, a “control system” may refer to software and/or hardwarethat is implemented to receive conditions parameters and respond byperforming calculations and presenting data to the user and/or changinghardware state or configuration in response to the data to reach arequired state.

As used herein, a “pumping system” may refer to an isocratic meteringpump or syringe pump or equivalent thereof used to pump various fluidsin the continuous manufacturing system.

As used herein, a “pumping buffer” may refer to a water-based solutioncontaining salts that are used to perform enzyme-based reactions.Examples include, but are not limited to, protein buffer solution (PBS)or sodium acetate buffer.

The size of an individual reactor may vary in length, outside diameterand internal diameter depending on the desired throughput. Anillustrative embodiment may be an individual reactor having a lengthbetween 1.5 inches and 14.5 inches or greater, an outside diameter (OD)between 0.125 inches and 0.75 inches or greater, or an internal diameter(ID) between 0.055 inches and 0.652 inches or greater, or somecombination thereof. Other embodiments are contemplated based upon thedesired throughput of the reactor. Other sizes of individual reactorsmay be used in accordance with the platforms described herein.

The reactor housing may be manufactured from any material havingsuitable properties, such as durability, strength, inertness towardreactor contents, etc. In some embodiments, the housing of theindividual reactor may be made from 6061 aluminum or similar material.

The reactor may be manufactured from any material having suitableproperties, such as durability, strength, inertness toward reactorcontents, etc. In some embodiments, the individual reactor may made of304 stainless steel cylindrical tubing or a similar material.

As used herein, “reactor conditions” may refer to conditions that ensurethe enzyme-resin complex(es) remain active to convert substrate toproduct and/or conditions that ensure the substrate and the product arestable and/or conditions that are optimal for enzyme reactions.Non-limiting examples of conditions the reactor may control include, butare not limited to, temperature, pressure, throughput volume,solvent(s), pH, oxygen level, other gas level(s), or combinationsthereof.

As used herein, a “graphical user interface (GUI)” may refer to a visualmethod of interacting with and/or controlling the continuousmanufacturing system from a computer including but not limited to textboxes and buttons within a software application.

As used herein, “reaction medium” may refer to a solution that is flowedthrough the reactor that contains the chemicals required to perform theenzyme-controlled reaction. This typically includes, but is not limitedto, a substrate (i.e., starting material), cofactor, gas, buffer salts,and other solvents. The chemical reaction takes place in the reactionmedium.

As used herein, “equilibrium buffer” may refer to a buffer that has thesame composition as the “reactor buffer” or “substrate solution,” butdevoid (or nearly devoid) of the substrate.

As used herein, “substrate solution” may refer to a solution that isflowed through the reactor that contains the chemicals required toperform the enzyme-controlled reaction. This may include one or more ofthe following: one or more substrates (starting material), one or morecofactors, one or more gases, one or more buffer salts, and one or moresolvent(s).

In some embodiments, the time for the conversion of the substrate toproduct may be varied to optimize the throughput and yield of thereaction. In some embodiments, the conversion of the substrate toproduct may proceed between 0.01 hours and 10 hours. In someembodiments, the conversion of the substrate to product may proceedbetween 0.01 hours and 100 hours, e.g., between about 10 hours and 100hours. In some embodiments, the conversion of the substrate to productmay proceed between about 0.01 hours and 1,000 hours, or between about10 hours and 1,000 hours or between about 100 hours and 1,000 hours. Insome embodiments, the conversion of the substrate to product may proceedmore than 1,000 hours.

As used herein, “flow rate” may refer to the rate at which a fluid ispassing through a reactor and can be measured by a flowmeter insertedprior and/or after an individual reactor.

In some embodiments, the flow rate of an individual reactor may bevaried to optimize the throughput and yield of the reaction. In someembodiments, the flow rate may range from about 0.1 μL/min to 1000μL/min, or about 0.1 mL/min to 100 mL/min, or about 0.1 μL/min to 10μL/min, or about 0.1 μL/min to 1 μL/min, or about 1 μL/min to 1000μL/min, or about 1 mL/min to 100 mL/min, or about 1 μL/min to 10 μL/min,or about 10 μL/min to 1000 μL/min, or about 10 mL/min to 100 mL/min, orabout 100 μL/min to 1000 μL/min. In some embodiments, the flow rate mayrange from about 10 μL/min to 100 μL/min. In other embodiments, the flowrate may range from about 100 μL/min to 1 mL/min. In furtherembodiments, the flow rate may range from about 1 mL/min to 10 mL/min.In some embodiments, the flow rate may range from about 1 mL/min to 1000mL/min, or about 1 mL/min to 100 mL/min, or about 1 mL/min to 10 mL/min,or 10 mL/min to 1000 mL/min, or about 10 mL/min to 100 mL/min, or about100 mL/min to 1000 mL/min. In some embodiments, the flow rate may rangefrom about 100 mL/min to 1 L/min. In some embodiments, the flow rate mayrange from about 1 L/min to 10 L/min. In some embodiments, the flow ratemay range from about 10 L/min to 100 L/min. In other embodiments theflow rate may range from about 1 L/min to 1000 L/min, or about 1 L/minto 100 L/min, or about 1 L/min to 10 L/min, or about 10 L/min to 1000L/min, or about 10 L/min to 100 L/min, or about 100 L/min to 1000 L/min.In further embodiments, the flow rate may be greater than 100 L/min.

As used herein, “residence time” may refer to the length of time a unitof fluid is inside a reactor.

In other embodiments, the residence time may be varied to optimize thethroughput and yield of a reaction. In some embodiments, the residencetime may range from about 0.1 minutes to 1 minutes, about 0.1 minutes to10 minutes, or about 0.1 minutes to 100 minutes, or about 1.0 minutes to100 minutes. In other embodiments, the residence time may range fromabout 0.5 minutes to 10 minutes, or about 0.5 minutes to 100 minutes, orabout 0.5 minutes to 1000 minutes. In some embodiments, the residencetime may range from about 1 minute to 10 minutes, or about 1 minutes to100 minutes, or about 1 minute to 1000 minutes. In other embodiments,the residence time may range from about 10 minutes to 100 minutes, orabout 10 minutes to 1000 minutes. In some embodiments, the residencetime may range from about 100 minutes to 1000 minutes. In someembodiments, the residence time may be greater than 100 minutes.

In some embodiments, the cell free manufacturing platform comprises acylindrical tube interior of the individual reactor comprising a resin.In some embodiments, the amount of resin packed inside (i.e., in thecylindrical tube interior) an individual reactor may vary. In someembodiments, an individual reactor may have about 0.01 g and 1.0 g ofresin packed inside. In some embodiments, an individual reactor may haveabout 0.1 g and 1.0 g of resin packed inside. In some embodiments, anindividual reactor may have about 1.0 g and 10 g of resin packed inside.In some embodiments, an individual reactor may have about 10 g and 100 gof resin packed inside. In some embodiments, an individual reactor mayhave about 100 g and 1.0 kg of resin packed inside. In some embodiments,an individual reactor may have about 1.0 kg and 10 kg of resin packedinside. In some embodiments, an individual reactor may have about 10 kgand 100 kg of resin packed inside. In some embodiments, an individualreactor may have more than 100 kg of resin packed inside.

In some embodiments, the amount of resin packed inside an individualreactor may be about 0.01 g to 1000 g, or about 0.01 g to 100 g, orabout 0.01 g to 10 g, or about 0.01 g to 1 g, or about 0.01 g to 0.1 g,or about 0.1 g to 1000 g, or about 0.1 g to 100 g, or about 0.1 g to 10g, or about 0.1 g to 1 g, or about 1 g to 1000 g, or about 1 g to 100 g,or about 1 g to 10 g, or about 10 g to 1000 g, or about 10 g to 100 g,or about 100 g to 1000 g. In other embodiments, the amount of resinpacked inside an individual reactor may be about 1 kg to 1000 kg, orabout 1 kg to 100 kg, or about 1 kg to 10 kg, or about 10 kg to 1000 kg,or about 10 kg to 100 kg, or about 100 kg to 1000 kg.

In some embodiments, the cell free manufacturing platform comprises acylindrical tube interior of the individual reactor comprising anenzyme. In some embodiments, the amount of enzyme inside (i.e., in thecylindrical tube interior) an individual reactor may vary. In someembodiments, an individual reactor may have about 0.01 mg to 1000 mg ofenzyme inside, or about 0.01 mg to 100 mg of enzyme inside, about 0.01mg to 10 mg of enzyme inside, about 0.01 mg to 1 mg of enzyme inside,about 0.01 mg to 0.1 mg of enzyme inside. In other embodiments,individual reactor may have about 0.1 mg to 1000 mg of enzyme inside, orabout 0.1 mg to 100 mg of enzyme inside, about 0.1 mg to 10 mg of enzymeinside, about 0.1 mg to 1 mg of enzyme inside. In further embodiments,individual reactor may have about 1 mg to 1000 mg of enzyme inside, orabout 1 mg to 100 mg of enzyme inside, about 1 mg to 10 mg of enzymeinside. In some embodiments, an individual reactor may have about 10 mgto 1000 mg of enzyme inside, or about 10 mg to 100 mg of enzyme inside.In other embodiments, an individual reactor may have about 100 mg to 1 gof enzyme inside. In some embodiments, an individual reactor may haveabout 1 g to 1000 g of enzyme inside, or about 1 g to 100 g of enzymeinside, or about 1 g to 10 g of enzyme inside. In other embodiments, anindividual reactor may have about 10 g to 1000 g of enzyme inside, orabout 10 g to 100 g of enzyme inside. In some embodiments, an individualreactor may have about 100 g to 1 kg of enzyme inside. In someembodiments, an individual reactor may have about 1 kg to 1000 kg ofenzyme inside, or about 1 kg to 100 kg of enzyme inside, or about 1 kgto 10 kg of enzyme inside. In other embodiments, an individual reactormay have about 10 kg to 1000 kg of enzyme inside, or about 10 kg to 100kg of enzyme inside. In some embodiments, an individual reactor may haveabout 100 kg to 1 Mg of enzyme inside. In some embodiments, anindividual reactor may have about 1 Mg to 1000 Mg of enzyme inside, orabout 1 Mg to 100 Mg of enzyme inside, or about 1 Mg to 10 Mg or enzymeinside. In other embodiments, an individual reactor may have about 10 Mgto 1000 Mg of enzyme inside, or about 10 Mg to 100 Mg of enzyme inside.In some embodiments, an individual reactor may have greater than 100 Mgof enzyme inside.

In some embodiments, the temperature of an individual reactor may bevaried to optimize the throughput and yield of the reaction. In someembodiments, the temperature of an individual reactor is varied with atemperature altering element. In some embodiments, the temperaturealtering element is attached to an individual reactor housing or anindividual reactor. In other embodiments, the temperature alteringelement is attaching to a cylindrical tube exterior. In someembodiments, the temperature of an individual reactor may be about 10°C. to 70° C., or about 10° C. to 60° C., or about 10° C. to 50° C., orabout 10° C. to 40° C., or about 10° C. to 30° C., or about 10° C. to20° C. In other embodiments, the temperature of an individual reactormay be about 20° C. to 70° C., or about 20° C. to 60° C., or about 20°C. to 50° C., or about 20° C. to 40° C., or about 20° C. to 30° C. Insome embodiments, the temperature of an individual reactor may be about30° C. to 70° C., or about 30° C. to 60° C., or about 30° C. to 50° C.,or about 30° C. to 40° C. In other embodiments, the temperature of anindividual reactor may be about 40° C. to 70° C., or about 40° C. to 60°C., or about 40° C. to 50° C. In some embodiments, the temperature of anindividual reactor may be about 50° C. to 70° C., or about 50° C. to 60°C. In some embodiments, the temperature of an individual reactor may bebetween 60° C. and 70° C. In some embodiments, the temperature of anindividual reactor may be greater than 70° C.

The temperature within an individual reactor may be measured at variousintervals. In some embodiments, the temperature within an individualreactor may be measured by a temperature sensor. In other embodiments,the temperature within an individual reactor may be measured by atemperature sensor within the cylindrical tube interior. For example,the temperature of an individual reactor may be measured once persecond. In some embodiments, the temperature of an individual reactormay be measured less than once per second. In other embodiments, thetemperature of an individual reactor is measured more than once persecond.

In some embodiments, the pH of an individual reactor (or of a solutiontherein) may be varied to optimize the throughput and yield of thereaction. In some embodiments, the pH of an individual reactor (or of asolution therein) may be varied using a pH measurement module adapted tointroduce an acid or a base into the solution within the individualreactor. In some embodiments, the pH of an individual reactor may beabout 4.0 to 10.0, or about 4.0 to 9.0, or about 4.0 to 8.0, or about4.0 to 7.0, or about 4.0 to 6.0, or about 4.0 to 5.0. In someembodiments, the pH of an individual reactor may be about 5.0 to 10.0,or about 5.0 to 9.0, or 5.0 to 8.0, or about 5.0 to 7.0, or about 5.0 to6.0. In some embodiments, the pH of an individual reactor may be about6.0 to 10, or about 6.0 to 9.0, or about 6.0 to 8.0, or about 6.0 to7.0. In some embodiments, the pH of an individual reactor may be about7.0 to 10.0, or about 7.0 to 9.0, or about 7.0 to 8.0. In someembodiments, the pH of an individual reactor may be about 8.0 to 10.0 orabout 8.0 to 9.0. In some embodiments, the pH of an individual reactormay be about 9.0 to 10.0. In some embodiments, the pH may be less than4.0. In some embodiments, the pH may be greater than 10.0

The pH in an individual reactor (or of a solution therein) may bemeasured at various intervals. In some embodiments, the pH within anindividual reactor (or within a solution therein) may be measured by apH sensor. In other embodiments, the pH within an individual reactor (orwithin a solution therein) may be measured by pH sensor within thecylindrical tube interior. In preferred embodiments, the pH of anindividual reactor (or of a solution therein) may be measured once persecond. In some embodiments, the pH of an individual reactor (or of asolution therein) may be measured less than once per second. In otherembodiments, the pH of an individual reactor (or of a solution therein)may be measured more than once per second. In some embodiments, the pHof an individual reactor may be changed through the addition of acidicor basic solutions.

In some embodiments, the pressure of an individual reactor may be variedto optimize the throughput and yield of the reaction. In otherembodiments, the pressure variation of an individual reactor may be abyproduct of introducing gases into the individual reactor. In furtherembodiments, the pressure variation of an individual reactor may be abyproduct of removing gases from the individual reactor. In someembodiments, the pressure may be about 0 psig (pound-force per squareinch) to 500 psig, or about 0 psig to 250 psig, or about 0 psig to 100psig, or about 0 psig to 50 psig, or about 0 psig to 10 psig, or about 0psig to 1 psig, or about 0 psig to 0.1 psig, or about 0 psig to 0.01psig. In other embodiments, the pressure may be about 0.01 psig to 500psig, or about 0.01 psig to 250 psig, or about 0.01 psig to 100 psig, orabout 0.01 psig to 50 psig, or about 0.01 psig to 10 psig, or about 0.01psig to 1 psig, or about 0.01 psig to 0.1 psig. In some embodiments, thepressure may be about 0.1 psig to 500 psig, or about 0.1 psig to 250psig, or about 0.1 psig to 100 psig, or about 0.1 psig to 50 psig, orabout 0.1 psig to 10 psig, or about 0.1 psig to 1 psig. In otherembodiments, the pressure may be about 10 psig to 500 psig, or about 10psig to 250 psig, or about 10 psig to 100 psig, or about 10 psig to 50psig. In some embodiments, the pressure may be about 50 psig to 500psig, or about 50 psig to 250 psig, or about 50 psig to 100 psig. Insome embodiments, the pressure may be about 100 psig to 250 psig, orabout 100 psig to 500 psig. In other embodiments, the pressure may beabout 250 psig to 500 psig. In further embodiments, the pressure may begreater than 500 psig.

The pressure within an individual reactor may be measured at variousintervals. In some embodiments, the pressure within an individualreactor may be measured by a pressure sensor. In other embodiments, thepressure within an individual reactor may be measured by pressure sensorwithin the cylindrical tube interior. In some embodiments, the pressureof an individual reactor may be measured once per second. In someembodiments, the pressure of an individual reactor may be measured lessthan once per second. In other embodiments, the pressure of anindividual reactor may be measured more than once per second.

In some embodiments, the amount of dissolved oxygen in an individualreactor may vary. In other embodiments, the amount of dissolved oxygenin a solution within an individual reactor may vary. In someembodiments, the amount of dissolved oxygen may be varied using a gasaddition module adapted to introduce gas into the solution within theindividual reactor. In other embodiments, the amount of dissolved oxygenmay be varied using a gas addition module adapted to introduce gas intothe cylindrical tubing interior of the individual reactor. In otherembodiments, the amount of dissolved oxygen may be varied using adegassing module adapted to remove gasses from the solution. In furtherembodiments, the amount of dissolved oxygen may be varied using adegassing module adapted to remove gasses from the cylindrical tubinginterior of the individual reactor. In some embodiments, the amount ofdissolved oxygen of an individual reactor may be about 0.0 ppm (partsper million) to 10 ppm, or about 0.0 ppm to 9.0 ppm, or about 0.0 ppm orabout 8.0 ppm, or about 0.0 ppm to 7.0 ppm, or about 0.0 ppm to 6.0 ppm,or about 0.0 ppm to 5.0 ppm, or about 0.0 ppm to 4.0 ppm, or about 0.0ppm to 3.0 ppm, or about 0.0 ppm to 2.0 ppm, or about 0.0 ppm to 1.0ppm. In some embodiments, the amount of dissolved oxygen of anindividual reactor may be about 1.0 ppm 10 ppm, or about 1.0 ppm to 9.0ppm, or about 1.0 ppm or about 8.0 ppm, or about 1.0 ppm to 7.0 ppm, orabout 1.0 ppm to 6.0 ppm, or about 1.0 ppm to 5.0 ppm, or about 1.0 ppmto 4.0 ppm, or about 1.0 ppm to 3.0 ppm, or about 1.0 ppm to 2.0 ppm. Inother embodiments, the amount of dissolved oxygen of an individualreactor may be about 2.0 ppm to 10 ppm, or about 2.0 ppm to 9.0 ppm, orabout 2.0 ppm or about 8.0 ppm, or about 2.0 ppm to 7.0 ppm, or about2.0 ppm to 6.0 ppm, or about 2.0 ppm to 5.0 ppm, or about 2.0 ppm to 4.0ppm, or about 2.0 ppm to 3.0 ppm. In some embodiments, the amount ofdissolved oxygen of an individual reactor may be about 3.0 ppm to 10ppm, or about 3.0 ppm to 9.0 ppm, or about 3.0 ppm or about 8.0 ppm, orabout 3.0 ppm to 7.0 ppm, or about 3.0 ppm to 6.0 ppm, or about 3.0 ppmto 5.0 ppm, or about 3.0 ppm to 4.0 ppm. In other embodiments, theamount of dissolved oxygen of an individual reactor may be about 4.0 ppmto 10 ppm, or about 4.0 ppm to 9.0 ppm, or about 4.0 ppm or about 8.0ppm, or about 4.0 ppm to 7.0 ppm, or about 4.0 ppm to 6.0 ppm, or about4.0 ppm to 5.0 ppm. In other embodiments, the amount of dissolved oxygenof an individual reactor may be about 5.0 ppm 10 ppm, or about 5.0 ppmto 9.0 ppm, or about 5.0 ppm or about 8.0 ppm, or about 5.0 ppm to 7.0ppm, or about 5.0 ppm to 6.0 ppm. In some embodiments, the amount ofdissolved oxygen of an individual reactor may be about 6.0 ppm to 10ppm, or about 6.0 ppm to 9.0 ppm, or about 6.0 ppm or about 8.0 ppm, orabout 6.0 ppm to 7.0 ppm. In some embodiments, the amount of dissolvedoxygen of an individual reactor may be about 7.0 ppm to 10 ppm, or about7.0 ppm to 9.0 ppm, or about 7.0 ppm or about 8.0 ppm. In otherembodiments, the amount of dissolved oxygen of an individual reactor maybe between 8.0 ppm to 10 ppm, or about 8.0 ppm to 9.0 ppm. In someembodiments, the amount of dissolved oxygen of an individual reactor maybe between 9.0 ppm to 10.0 ppm. In further embodiments, the amount ofdissolved oxygen in an individual reactor is greater than 10.0 ppm.

The amount of dissolved oxygen within an individual reactor (or within asolution therein) may be measured at various intervals. In someembodiments, the amount of dissolved oxygen within an individual reactor(or within a solution therein) may be measured by a dissolved oxygen(DO) sensor. In other embodiments, the amount of dissolved oxygen withinan individual reactor (or within a solution therein) may be measured bya dissolved oxygen (DO) sensor within the cylindrical tube interior. Insome embodiments, the amount of dissolved oxygen of an individualreactor (or of a solution therein) may be measured once per second. Insome embodiments, the amount of dissolved oxygen of an individualreactor (or of a solution therein) may be measured less than once persecond. In other embodiments, the amount of dissolved oxygen of anindividual reactor (or of a solution therein) may be measured more thanonce per second.

In some embodiments, dissolved oxygen may be removed via adeoxygenation/degassing machine (i.e., deoxygenation/degassing module).As used herein, a “deoxygenation machine” or “degassing machine” or“deoxygenation module” or “degassing module” may refer to a device whichremoves the amount of dissolved oxygen and/or other gasses (e.g.,nitrogen) in a fluid (i.e., a solution) when the fluid is flowed throughthe device. In other embodiments, nitrogen gas is introduced to anindividual reactor to reduce the levels of oxygen.

As used herein, a “gassing machine” or “gassing module” may refer to adevice which adds an amount of dissolved oxygen and/or other gasses(e.g., nitrogen) into a fluid (i.e., a solution) when the fluid isflowed through the device. In some embodiments, the cell manufacturingplatform comprises a gas addition module adapted to introduce gas intothe solution. In some embodiments, oxygen, nitrogen or a combinationthereof may be added to an individual reactor. Other gases may be addedor removed from an individual reactor in accordance with the platformdescribed herein.

As used herein, a “chemical stream” may refer to a solution thatcontains a substrate, product, intermediate, cofactor or anotherchemical.

Referring now to FIGS. 1-34B, the present invention features a cell freemanufacturing platform for continuous chemical production.

The present invention features a cell-free manufacturing platform forchemical production. In some embodiments, the platform comprises one ormore individual reactors and a pumping system adapted to flow a solutionthrough the one or more individual reactors. In some embodiments, eachof the one or more individual reactors comprises a cylindrical tubecomprising a first end and a second end. In some embodiments, both thefirst end of the cylindrical tube and the second end of the cylindricaltube comprise fittings (i.e., stainless steel fittings). In someembodiments, a cylindrical tube interior of the individual reactorcomprises a resin and an enzyme. In some embodiments, each of the one ormore individual reactors has an input tubing connected at the first endof the cylindrical tube and an output tubing connected at the second endof the cylindrical tube. In some embodiments, the cell-freemanufacturing platform is able to automatically change each of the oneor more reactors conditions based on input from the sensors.

In some embodiments, the cell-free manufacturing platform describedherein is a closed system. In other embodiments, the cell-freemanufacturing platform described herein is an open system.

In some embodiments, each of the one or more individual reactorscomprises an individual reactor housing. In some embodiments, theindividual reactor housing surrounds and is fastened to the individualreactor. In embodiments, the cell-free manufacturing platform furthercomprises a temperature altering element attached to the individualreactor housing or the individual reactor. In some embodiments, thetemperature altering element is a thermoelectric cooler (TEC). In someembodiments, the temperature altering element is a flexible heatingelement.

In some embodiments, the cell-free manufacturing platform furthercomprises a spectrometer attached in series with the one or moreindividual reactors. In other embodiments, the cell-free manufacturingplatform further comprises a degassing module adapted to remove gassesfrom the solution. In some embodiments, the degassing module is adeoxygenation module. In some embodiments, the deoxygenation module isadapted to remove oxygen from the solution. In some embodiments, thecell-free manufacturing platform further comprises a gas addition moduleadapted to introduce gas into the solution. In other embodiments, thecell-free manufacturing platform further comprises a pH module adaptedto introduce an acid or base into the solution. In some embodiments, thecell-free manufacturing platform further comprises a graphical userinterface (GUI) adapted to control automation software and hardware.

In some embodiments, present invention features a cell-freemanufacturing platform for chemical production. In some embodiments, theplatform comprises one or more individual reactors. In some embodiments,each of the one or more reactors comprises a cylindrical tube withstainless steel fittings at both ends. In other embodiments, each of theone or more reactors comprises a resin, an enzyme, and one or moresensors. In some embodiments, the platform comprises an individualreactor housing. In some embodiments, the housing surrounds and isfastened to the individual reactor. In some embodiments, the platformcomprises a temperature altering element (e.g., a thermoelectric cooler(TEC)) attached to the reactor housing or the individual reactor. Insome embodiments, the platform comprises a pumping system adapted toflow a solution through the one or more reactors. In some embodiments,the platform comprises a degassing module (e.g., a deoxygenation module)adapted to remove gasses from the solution. In some embodiments, theplatform comprises a gas addition module adapted to introduce gas intothe solution. In some embodiments, the platform comprises a spectrometerattached in series with the reactor(s). In some embodiments, theplatform comprises a graphical user interface (GUI). In someembodiments, the platform comprises an automation software and hardware.In some embodiments, the GUI is adapted to control the automationsoftware and hardware. In some embodiments, the individual reactor hasinput, and output tubing connected at each end of the cylindrical tubeto create a closed system. In some embodiments, the platform is able toautomatically change each of the one or more reactors conditions basedon input from the sensors.

In other embodiments, the present invention may feature a cell-freecontinuous manufacturing platform for chemical production. In someembodiments, the platform comprises an individual reactor comprising acylindrical tube with stainless steel fittings at both ends of areactor. In some embodiments, the platform comprises a plurality ofreactors connected in series. In other embodiments, the platformcomprises a plurality of reactors in parallel. In some embodiments, thereactor comprises resin, enzymes, and sensors. In some embodiments, theplatform comprises an individual or multiple reactor housing, whereinthe housing surrounds and is fastened to the individual reactor(s). Insome embodiments, the platform comprises a combination of thermoelectriccoolers (TEC) or other temperature altering element(s) attached to thehousing(s), a pumping system, a deoxygenation and/or degassing module, agas addition module, a graphical user interface (GUI), and an automationsoftware and hardware the device is ran on. In some embodiments, theindividual reactor is sealed at each end of the cylindrical tube tocreate a closed system. In some embodiments, the platform is able toautomatically change the reactor conditions based on input from thesensors. In some embodiments, the platform comprises a spectrometer tomeasure wavelength and absorption of volume entering and exiting. Inother embodiments, the platform comprises a spectrometer to measureoutput color wavelength and/or absorption. In further embodiments, theplatform is able to automatically change the reactor conditions based oninput from the sensors.

In some embodiments, the one or more individual reactors are connectedin parallel. In other embodiments, at least two of the one or moreindividual reactors are connected in parallel. In some embodiments, morethan one reactor is connected in parallel. In further embodiments, theone or more individual reactors connected in parallel are connected to asingle pump. In some embodiments, the parallel reactors are connected toa single pump. In some embodiments, the flow through the parallelreactors is controlled via software and control valves.

In some embodiments, the enzymes are chosen from a group consisting of:MBP-Aldo (Aldo), Dihydroxy Acid Dehydratase (TvDHAD), Pyruvate Oxidase(PyOx), Acetyl-phosphate transferase (PTA), Acetyl-CoA acetyltransferase(PhaA), HMG-CoA Synthase A110G (HMGS), HMG-CoA Reductase (HMGR),Mevalonate Kinase (MVK), Phosphomevalonate Kinase (PMVK),Diphosphomevalonate Kinase (MDC), Isopentyl-PP Isomerase (IDI),Farnesyl-PP synthase S82F (FPPS), and Prenyl transferase (NphB). In someembodiments, the enzymes comprise MBP-Aldo (Aldo), Dihydroxy AcidDehydratase (TvDHAD), Pyruvate Oxidase (PyOx), Acetyl-phosphatetransferase (PTA), Acetyl-CoA acetyltransferase (PhaA), HMG-CoA SynthaseA110G (HMGS), HMG-CoA Reductase (HMGR), Mevalonate Kinase (MVK),Phosphomevalonate Kinase (PMVK), Diphosphomevalonate Kinase (MDC),Isopentyl-PP Isomerase (IDI), Farnesyl-PP synthase S82F (FPPS), Prenyltransferase (NphB), or a combination thereof.

In some embodiments, the enzymes are immobilized. In other embodiments,the enzymes are non-immobilized.

In some embodiments, the plurality of sensors comprises a temperaturesensor. In some embodiments, the plurality of sensors comprises a pHsensor. In some embodiments, the plurality of sensors comprises apressure sensor. In some embodiments, the plurality of sensors comprisesa flow rate sensor. In some embodiments, the plurality of sensorscomprises a dissolved oxygen (DO) sensor. In some embodiments, theplurality of sensors comprises a spectrometer. In some embodiments, thecylindrical tube interior of the individual reactor further comprisesone or more sensors. In some embodiments, the one or more sensorscomprises a temperature sensor, a pH sensor, a pressure sensor, a flowrate sensor, a dissolved oxygen (DO) sensor, a spectrometer, or acombination thereof.

In some embodiments, an individual reactor has a percent yield of about10-100%. In some embodiments, an individual reactor has a percent yieldof about 10%. In some embodiments, an individual reactor has a percentyield of about 20%. In some embodiments, an individual reactor has apercent yield of about 30%. In some embodiments, an individual reactorhas a percent yield of about 40%. In some embodiments, an individualreactor has a percent yield of about 50%. In some embodiments, anindividual reactor has a percent yield of about 60%. In someembodiments, an individual reactor has a percent yield of about 70%. Insome embodiments, an individual reactor has a percent yield of about80%. In some embodiments, an individual reactor has a percent yield ofabout 90%. In some embodiments, an individual reactor has a percentyield of about 95%. In some embodiments, an individual reactor has apercent yield of about 98%. In some embodiments, an individual reactorhas a percent yield of about 99%. In some embodiments, an individualreactor has a percent yield of about 100%.

In some embodiments, each individual reactor allows an enzyme-resincomplex to be contained within the reactor. In some embodiments, thereactor contains a single enzyme-resin complex. In other embodiments,the reactor contains multiple enzyme-resin complexes.

In some embodiments, each individual reactor contains a set amount ofenzyme-resin complex. In other embodiments, a set amount of enzyme resincomplex may allow for a tunable concentration of enzyme can be achievedin each individual reactor. For example, if more enzyme is desired, theindividual reactor may be packed with more enzyme-resin complex, andvice versa.

In some embodiments, each individual reactor provides the necessaryreactor conditions to ensure that the enzyme-resin complex(es) remainactive to convert substrate to product. In some embodiments, the reactorconditions are finely controlled to ensure the lifetime of the enzyme.Non-limiting examples of conditions the reactor may control include butare not limited to temperature or pH or oxygen level.

In some embodiments, each individual reactor has customizabletemperature control to ensure each enzyme obtains its optimal reactiontemperature. In some embodiments, each individual reactor has theability to control reactor temperature within +/−0.1° C. by using thegraphical user interface (GUI). There is no manual readjustment to thesystem, only algorithmic feedback, and control after user temperaturesetpoint to a computer.

Without wishing to limit the present invention to any theory ormechanism it is believed that temperature is highly important for anyenzymes during a reaction. For example, Aldo (that converts glycerolinto glyceric acid) requires an optimal temperature of 37° C. (19.6 mM,98% yield) however, the next enzyme in the pathway (DHAD) requires 45°C. to convert glyceric acid into pyruvic acid (6.4 mM, 32% yield, Table1). If these reaction temperatures are not adhered to, reaction yieldand rate suffer. FIG. 2 shows that 45° C. is optimal for DHAD, if thereaction temperature drops to 32° C. or increases to 55° C. then asignificant move from optimal conversion is observed.

In some embodiments, each individual reactor has reactor fluid pHadjustment and control to afford the unique pH requirements for eachenzyme (Table 1). In some embodiments, the pH sensor and automatedfeedback loops to ensure that the pH of the reaction medium can bechanged between individual continuous reactors. In some embodiments, tochange the pH of the reaction medium a certain volume of a certain pHsolution is injected into the reactor system to change and maintain aspecific pH. This allows each reactor to have optimal pH to avoidprotein precipitation, loss of reactivity, or other degradinginfluences.

In some embodiments, the reactor system has the ability to add gases toindividual reactors for enzymes that require a gas. Non-limitingexamples may include oxygen or nitrogen. As described herein, oxygen andnitrogen addition to the reactors have been demonstrated to ensureeffective enzyme transformations. This was crucial, as without oxygenaddition, immobilized ALDO afforded 0% product. However, afterintroduction of oxygen into the reactor, 98% yield was obtained (19.6mM).

In some embodiments, the reactor system can reduce the oxygen present inthe reaction medium when required. In some embodiments, oxygen isremoved through the use of sonication and a vacuum. In some embodiments,oxygen is removed through the addition of nitrogen.

As described herein, the DHAD enzyme evaluated required oxygen removalto increase reaction efficiency. A module for this system was introducedthat removed the oxygen present in the fluid down to 4.07 ppm throughthe use of sonication and a vacuum. This device allows removal of pO₂from 7.50 ppm to 4.07 ppm in 10 minutes at room temperature. In additionto the module, nitrogen gas can be introduced to further reduce theamount of oxygen in the medium.

In some embodiments, each individual reactor allows a starting material(substrate solution) to be pumped through the reactor using a standardlaboratory pump. The substrate solution is injected into the reactor,the solution moves through the reactor to interact with the enzyme andcause a chemical reaction. Once the chemical reaction is complete, thereacted fluid moves out of the reactor to a collection flask.

In some embodiments, the reactor can operate in a continuous mode,wherein the pump injecting the fluid into the individual reactor wouldalways be pumping fluid. In other embodiments, the reactor can operatein a semi-continuous mode, wherein the pump injects the fluid into theindividual reactor for a certain amount of time and then stops for acertain amount of time to hold the fluid in the reactor. In someembodiments, after the set period of time, the pump restarts pushing thereacted fluid out of the reactor system. Without wish to limit thepresent invention to any theories or mechanisms, it is thought that asemi-continuous mode allows longer enzyme reaction times to beaccommodated.

In some embodiments, the reactor system may allow additional chemicalsolutions to be added to the continuous system between the individualreactors. In some embodiments, a computer-controlled pump allowschemical solutions to be injected into the continuous manufacturingsystem when instructed. Non-limited examples of chemical solutions mayinclude but are not limited to buffers, cofactors, and chemicalreagents. Examples of buffers may include, but are not limited to, 50 mMTris at pH 12.0 to adjust process flow pH from pH 7.7 to pH 8.5, 100 mMTris K₂HPO₄: KH₂PO₄ (1:1) at pH 6.33 in order to adjust process flowfrom pH 7.7 to pH 6.5, and 50 mM Tris at pH 12.85 in order to adjustprocess flow from pH 6.47 to pH 8.0.

In some embodiments, the platform contains a wide range of sensorsallowing full system automation. These sensors include, but are notlimited to temperature sensors/thermistors, pressure sensors,flowmeters, pH sensors/probes, dissolved oxygen sensors/probes, andspectrometers. Deviation away from a programmed optimal value in the GUIor code (temperature, flow rate, pH, pressure, etc.) triggers systemadjustment through an algorithmic feedback loop(s) and correspondingchanges in hardware state(s) to allow for system conditions to reach arequired state.

In some embodiments, the individual reactor is scalable. In someembodiments, the length, diameter, or number of individual reactors maybe increased to achieve a higher throughput. There is no theoreticallimit on the size of these reactors or the number. In some embodiments,the individual reactors can be different sizes, shapes, andconfigurations.

In some embodiments, the cylindrical tubing of an individual reactor maybe virtually any diameter tubing and of any length the user wants toallow for more or less resin/enzyme, or shorter/longer reaction times.In other embodiments, the fittings (e.g., stainless steel fittings) atthe ends of the cylindrical tubing of an individual reactor may becustomized as well to allow for various sizes of input/output tubing tobe used. In some embodiments, the material of the cylindrical tubing theindividual reactor is made out of can be changed to allow for moreoptimal thermal conductivity or other parameters if the reaction permitscoming into contact with the material.

In some embodiments, the reactor system has a customizable userinterface for users to control the reactor with a mouse click, commonlyknown as a GUI (graphical user interface).

EXAMPLE

The following is a non-limiting example of the present invention. It isto be understood that said example is not intended to limit the presentinvention in any way. Equivalents or substitutes are within the scope ofthe present invention.

1.1 Individual Reactor Design: The individual reactors are made of 304stainless steel cylindrical tubing. The individual reactor housings aremade of 6061 aluminum. The individual reactor housings are used as heattransfer vehicles to change the temperature of the reactors held withinby attaching thermoelectric coolers (TEC) elements to the housings (FIG.3). The temperature(s) of the reactor(s) can also be altered throughattaching flexible heating elements to the exterior of the reactor(s).The individual reactors can be heated and cooled from 12° C. and 55° C.Compared to a conventional batch reactor, these reactors can reach thedesired temperature in 11 minutes when tested at a set temperature of45° C. (FIG. 4). Each individual reactor is fitted with Swagelokfittings at each end of the 304 stainless steel cylindrical tubing toseal the tubing to create a reactor. Additionally, the Swagelok fittingsallow inlet and outlet tubing to be attached to the reactor to introduceand remove fluids from the individual reactor. To note, the volume,shape, size and material of the reactor can be changed, and differentmaterials such as plastics and different volumes including 1 g to 330 ghave been tested.

To ensure the individual reactors would comply with the pumping system,the pressure of the reactors was tested. Internal pressure of theindividual reactors was monitored in real time over 24 hours by pumpinga buffer (50 mM Tris, 20 mM glyceric Acid, pH 7.7) through the first tworeactors in the pathway. Buffer was pumped through the individualreactor containing resin at a temperature of 21° C. (i.e., roomtemperature) with a flow rate of 10 mL/min. After the first reactor,there is a joint where a buffer (50 mM Tris, pH 12.0) was pumped intothe process flow along with the Tris solution in order to modify its pHlevel. To monitor the pressure of the individual reactor in real time, apython program was written to retrieve the pressure level from both theprocess pumps once per second. In this testing system, the pumps aredirectly connected to the reactors through PFA tubing and thus thepressure calculated by the pumps will be a direct sampling of theinternal pressure of the individual reactors.

The calculated pressure inside the individual reactors ranged from 0-70psig. The average internal pressure of the first reactor in the systemwas <0.01 psig when tested over a 24-hour window (FIGS. 5A-5B). Theaverage internal pressure of the second reactor in the system was <0.1psig when tested over the same 24-hour window. This low pressure isrequired for scale up of the reactor and also indicates that the systemdoes not induce resin swelling through uptake of fluid. Finally, thepressure monitoring used in this system also allows the user to setpressure warning limits in the GUI for safety and control aspects. Thisfeeds into the automated control of the continuous manufacturing systemas a whole.

The amount of enzyme that a standard individual reactor can hold wasalso calculated. The individual reactors tested here can hold 57 g ofenzyme-resin complex (<5.5 g of isolated enzyme). This means that onereactor with a size of 14″ length (L)×0.652″ inner diameter (ID) (79 mLtotal volume, FIG. 6), can hold as much enzyme as a 1000 L fermenter orgreater when the enzyme expression level in a batch reactor is 5.5 mg/Lor greater. This is just one example and the comparison changes whenenzymes are expressed at different levels. However, this dramaticimprovement means that a 1 L individual reactor described herein couldhold the same amount of enzyme as a 12,600 L batch fermenter when theenzyme expression level in a batch reactor is 5.5 mg/L.

1.2 Temperature Control for the Individual Reactors: Individual reactortemperature control is achieved through aproportional-integral-derivative (PID) algorithm and supporting codecontained within an Arduino Mega 2560 microcontroller or STM32L476RGmicrocontroller or similar. The temperature sensors used are NTCthermistors that operate by a change in electrical resistance as theirtemperatures change. This change in resistance is relayed to themicrocontroller as a voltage through use of a voltage divider circuit(FIGS. 7A-7B). The thermistors are placed within indentions in thereactor housings and are held in place once the housings are fastenedclose. The thermistors contact both the reactor housing and the reactoritself. In the case of a reactor being heated with a flexible siliconheating element or similar, no reactor housing is used. In this case thethermistor is inserted between the exterior of the reactor and theheating element. The difference in temperature between where thethermistors are placed and inside the reactor has been measured and isaccounted for in the software. Within the PID algorithm software, thethermistors' resistance values are converted to an accurate temperaturereading by converting the incoming voltage reading back to an electricalresistance, and then is further calculated into degrees Kelvin by usinga simplified version of the Steinhart-Hart equation (FIG. 8). This valueis then converted from Kelvin to Celsius. The temperature of theindividual reactor(s) is read once per second. The temperature readingis fed into a PID loop that responds to the current temperature andmodifies the pulse width modulation (PWM) duty cycle of the electricaloutput that is powering the heating elements to change the temperatureof the individual reactor to the correct level. The circuitry can beseen in FIGS. 9A-9B. The PID software framework is licensed under theMIT permissive license. This software foundation has been edited andexpanded to control the system described herein. The PID loop has beenfine tuned to minimize the amount of temperature overshooting andequilibration time (FIG. 4). The TECs causing the temperature change areadhered to the individual reactor housings using a thermal conductiveglue. The glue keeps the TECs securely fastened to the housings whileallowing for heat transfer to continue uninterrupted (FIG. 3). Theflexible silicon heating elements or similar have adhesive on one sideto allow for attaching to various objects.

The temperature control system can also cool the individual reactors to12° C. (FIGS. 10A-10B), no change was observed in the performance.Additionally, the amount of power that was required to cool to thistemperature was 0.027 kW; this has a staggering benefit compared tolarge batch reactors that require large amounts of electricity to cool.Cooling can be achieved by flipping the TECs to their other side, as theother side of the TEC is the “cold” side. Alternatively, if there is anapplication where a reactor needs to be both heated and cooled, analternate circuit can be implemented that allows for heating/cooling tobe switched by inserting a separate wire into the correspondingmicrocontroller digital pulse width modulation (PWM) pin, instead of thewire used to activate heating (FIG. 9B). If cooling is required, it isnecessary to attach a heatsink apparatus onto the Peltier elements. Theelements work on a temperature differential between each side of theplate, so if the cold side is being used to cool down the reactor theother side will heat up. If no heatsink is used, the hot side willoverpower the cold side until an equilibrium temperature above ambientis reached. The heat sink keeps the “hot” side cooler, which allows forthe cold side to maintain a cooler temperature. A thermal image of thisoccurring can be seen in FIG. 10B.

1.3 The Individual Reactors have strict pH control: Between each reactorwill be an injection point where acidic or basic solution can becontinuously injected into the process flow to provide a change in pHthat matches the pH requirement for the next reactor in line. Thecorrect flow rate and pH level of the required injections have beendetermined through experimentation. Additionally, a pH sensor wasimplemented that can measure the pH of a flowing solution in real-timeto provide accurate pH monitoring. The pH sensor is connected in-lineand sends pH values to the control system serially once per second. Thisprovides the user(s) additional data on the accuracy of the pHadjustment as well as automatically changes the flowrate of the pHinjection in order to maintain an accurate pH output.

1.4 Gases can be added to the Individual Reactors: The system hasadditional injection points for gas injection into the fluid flow beforean individual reactor. The required gas tank is connected to the systemvia a mass flow controller. Here, the user can set a flow rate of gas toenter into the individual reactors in a continuous manner. The mass flowcontroller(s) are controlled via voltage signals. These signals can beadjusted both in an analog manner using a voltage divider circuit with apotentiometer (manual turning of a dial), or digitally via sendingdigital signals through a digital to analog converter (DAC). Three gaseshave been tested thus far: compressed air, oxygen, and nitrogen. Thecompressed air was used for the Aldo enzyme that requires supplementaloxygen to react properly, and compressed nitrogen can be used to reduceoxygen concentrations of the fluid. Gas tanks are connected to the massflow controller(s) via tubing and stainless-steel fittings. Mass flowcontrollers are connected to the individual reactors via tubing andSwagelok fittings. Mass flow controllers are programmed to allow aspecific volume of gas per minute to pass through the mass flowcontroller into the individual reactor. The correct ratio of gas tofluid has been experimentally tested and optimized and is roughly a 1:1volumetric ratio.

1.5 A module to decrease oxygen content in the fluid entering theindividual reactors: In addition to the nitrogen gas being added toreduce oxygen, the system utilizes a deoxygenation and/or degassingmachine to reduce the amount of oxygen within the process flow. Thedeoxygenation/degassing module will be placed in series with the flowand the fluid exiting the machine will have up to 3.68 ppm of oxygenremoved. A reduction of dissolved oxygen from 7.75 ppm to 4.07 ppm, areduction of almost 50% was shown (FIG. 11).

1.6 Injection of additional chemical streams into the individualreactors: In addition to the injection of solutions to adjust pH, therewill be injection points to add chemical streams to the process flow.These chemical streams are specific to the reactors they are associatedwith and will alter the chemistry required to yield a product asintended.

1.7 System control and automation on the Individual Reactors: The systemcontains various sensors to monitor and/or adapt reaction conditionswhen told to by the user or automatically. Sensors include, but are notlimited to, temperature sensors/thermistors, flowmeters, pressuresensors, pH probes, dissolved oxygen probes, mass flow controllers, andspectrometers. The temperature sensors for each individual reactor feeddata to the microcontroller which then autonomously adjusts the poweroutput to the heating elements, which alters the temperature of saidreactor. The flow meter monitors the rate at which the fluid within thesystem is flowing through the reactor and streams that data to themicrocontroller. If the process flow has slowed to a rate considered notoptimal, the microcontroller sends commands to incrementally increase ordecrease the flow rate of the pumps until an acceptable flow rate isachieved. The pH probe(s) monitor the pH of the volume passing across itand adjusts the amount of acidic or basic solution being injected intothe process flow to achieve the desired pH level. The dissolved oxygenprobe measures the amount of oxygen in the solution and themicrocontroller can adjust the rate at which oxygen or other gases arebeing added to the process flow via mass flow controller. The pressuresensors measure the pressure within the process flow. The spectrometermeasures the wavelength of light of the volume exiting the reactor andreads the intensity across the spectrum of 340.6 nm to 1010 nm and theabsorbance levels across the same spectrum of light.

1.8 Usage of Parallel Reactors with equal flow: The system can utilizereactors in a parallel fashion. Two individual reactors can be connectedto a single pump in parallel. The two reactors have achieved equal flowthrough each by implementing control software with accompanyinghardware. The accompanying hardware in this configuration is aflowmeter(s) and control valves. The control valves are placed upstreamfrom the reactors and the flowmeter(s) are placed downstream from thereactor in series. The software is connected to the flowmeter(s) andcalculates the flow rate of the output of each reactor connected inparallel. The software keeps a total of the amount flowed through eachreactor. The software then makes determinations based on the flow ratesand amounts flowed to temporarily stop or continue flow in individualreactors via solenoid valves to keep the total amount of solutionflowing through each to be even (FIG. 27)

1.9 System output color and absorption collection: The system contains aspectrometer in which system output wavelength and absorption can bemeasured. A flow cell is connected in series with system output throughwhich the output flows. A light source is connected to the flow cell viafiber optic cabling. The flow cell is connected to the spectrometer viafiber optic cabling. The spectrometer readings can be retrieved in twoways. One way is by using a commercial software package included withthe spectrometer in tandem with the spectrometer unit to read thewavelength of the color of the output or the absorption of the output inreal time (FIG. 28). The second way is through software that was writtento communicate with the spectrometer via RS232 to retrieve the data. Thesoftware can retrieve the sample's wavelength intensity over thespectrum from 340.6 nm to 1010 nm. The software also performscalculations to convert the intensity levels of the sample to anabsorbance value. The software has the capability to plot its findingson a graph and/or save them to a log file or spreadsheet document orsimilar (FIG. 29). The software was written in Python. The data from thesample's color and absorbance can be further analyzed to makepredictions of the output's concentration based on the color/spectraldata.

1.10 Reactors can be different sizes, shapes, and configurations:Individual reactors can be different diameters and lengths to allow fordifferent residence times for the fluid being flowed through thereactor. Currently, three different sizes of reactors have been tried:

-   -   1. The first size reactor is 13.5″ in length with a 0.25″        outside diameter (OD) and a 0.180″ inner diameter (ID) resulting        in a volume of 5.63 mL (Table 2)    -   2. The second size reactor is 14.5″ in length with a 0.75″ OD        and a 0.652″ (ID), resulting in a volume of 79.33 mL (Table 2).    -   3. The third size reactor is 5.5″ in length with a 0.25″ OD and        a 0.218″ ID, resulting in a volume of 1.64 mL (Table 2).

TABLE 2 Dimensions of the three sizes of reactors along with volume andresidence time data. Reactor #1 Reactor #2 Reactor #3 Outside diameter(in) 0.25 0.25 0.75 Inside Diameter (in) 0.180 0.218 0.652 Height (in)13.5 5.5 14 PSI max @ 72 F. 6125 2000 2190 Reactor Volume (in³) 0.3440.205 4.674 Reactor volume (cm³) 5.629 3.364 76.598 Resin volume (cm³)4.912 2.935 66.831 Volume for media (cm³) 0.718 0.429 9.766 Flow rate(mL/min) 0.01 0.01 0.01 Residence Time 71.776 42.892 976.619 (minutes)Residence Time (hours) 1.196 0.715 16.277

There were two different sizes of reactor housings made, one for the ¼″OD reactor and one for the ¾″ OD reactor. The complete dimensions can beseen on their drawings in FIGS. 12A-12D. To accommodate the shorterlength of the third reactor, a reactor housing meant for the ¼ ODreactor was cut to length. The reactor sizes and their calculatedresidence times can be seen in Table 2.

1.11 The reactor system has a user interface to control the reactor: Thesystem has an optional user interface to allow users to specify whattemperature each reactor should be programmed to, start and stop thepumps within the system, manually check the pressures each pump isunder, and set upper pressure limits on the pumps (FIGS. 9A-9B). The GUIwas written in Python using the Kivy framework.

1.12 The system has a graphical dashboard to monitor the variables foreach reactor in real time: In addition to the control the user interfacewill provide, there is a monitoring dashboard that displays eachreactor's temperature, the flowrate(s) as provided by the flowmeter(s),the pressure as seen by the pumps, and the PWM duty cycle being appliedto each Peltier element. The data will be displayed using Grafana andstored in a database using InfluxDB, in conjunction with NodeRED. Thisenvironment will be powered and hosted by a raspberry pi. The data flowcan be seen in FIG. 13.

1.13 Individual Reactors can be linked into a sequence to affordmulti-step enzyme reactors: Reactors may be connected in series via ¼″OD tubing to allow for multi-step enzyme reactions. The pathway requires13 different enzymes which translates to 13 different reactors. Thesereactors will be connected in series along with injection points for pHcontrol and gas addition.

Example 2.0 Experimental Examples

2.1 Production of glyceric acid from glycerol using immobilized alditoloxidase (Aldo) (FIG. 14): A reactor of 13.5″ in length with a 0.25″outside diameter and a 0.180″ internal diameter containing immobilizedAldo enzyme (240 mg enzyme on 4.00 g of resin) was heated to 37° C. andequilibrated for one hour with equilibration buffer (50 mM tris, pH 8.5)being passed through the reactor. After one hour had subsided, thesubstrate solution (40 mL, 50 mM Tris, pH 8.5 20 mM glycerol) was flowedthrough the reactor at a flow rate of 20 μL/min. Prior to entering thereactor, the substrate solution was mixed with an equal flow rate ofcompressed air (0.02 standard cubic centimeters per minute (sccm)) toyield a total flow rate through the reactor of 40 μL/min (18-minuteresidence time). After the solution had passed through the reactor, itwas collected in the same beaker as the starting solution to allow thesolution to recycle through the individual reactor. The reaction wasallowed to proceed for 72 hours with sampling performed every 24 hours.For sampling, 100 μl of the reaction fluid was examined on ahigh-performance liquid chromatography (HPLC) system to examine theamount of glycerol and glyceric acid. The HPLC method was as follows: AnAgilent 1200 HPLC was equipped with a 30 cm Aminex HPX-87H column and amicro-guard cation H-refill cartridge. The column was heated to 55° C.and the sample block was maintained at 25 ° C. For each sample, 1 μL wasinjected and an isocratic mobile phase comprised of 100% sulfuric acid(10 mM) was used. The sample run time was a total of 45 minutes withglyceric acid eluting at 17.2 mins and glycerol eluting at 21.0 minutes.For detection, a RID detector (Agilent) was used after a 2 hequilibration period produced a stable baseline. Upon analysis, thefollowing data was obtained; 24 h reaction time=21% glyceric acid and79% glycerol, 48 h reaction time=76% glyceric acid and 24% glycerol, 72h=98% glyceric acid, 3% glycerol. Upon 72 h the yield of the reactionconverting glycerol to glyceric acid was 99% (19.6 mM, 2.06 g/L). Tonote, the same reaction was carried out without addition of compressedair and 0% conversion was observed. This reaction was scaled to areactor with dimensions 14″ length×0.75″ outside diameter and 0.652″inner diameter (Example 2.13 below). 98% conversion to product wasobserved (19.6 mM glyceric acid) in 66 hours under the same conditionsas described above.

2.2 Production of pyruvic acid from glyceric acid using immobilizeddihydroxy acid dehydratase (DHAD) (FIG. 15): A reactor of 13.5″ inlength with a 0.25″ outside diameter and a 0.180″ internal diametercontaining immobilized DHAD enzyme (360 mg on 3.70 g of resin) washeated to 55° C. and allowed to equilibrate for one hour while passingequilibration buffer (250 mM HEPES, pH 7.4, 2.5 mM MgCl₂.6H₂O) throughthe reactor. After one hour had subsided, the substrate solution (40 mL,250 mM HEPES, pH 7.4, 2.5 mM MgCl₂.6H₂O, 20 mM glyceric acid) was flowedthrough the reactor at a flow rate of 10 μL/min (72 min residence time,FIG. 15). After the solution had passed through the reactor it wascollected in the same beaker as the starting solution to allow thesolution to recycle through the individual reactor. The reaction wasallowed to proceed for 48 hours with sampling performed every 24 hours.For sampling, 100 μl of the reaction fluid was examined on a HPLC systemto examine the amount of glyceric acid and pyruvic acid. The HPLC methodwas the same as above with pyruvic acid eluting at 15.8 mins andglyceric acid eluting at 17.2 minutes. Upon analysis, the following datawas obtained; 24 hours reactivity=17% pyruvic acid and 48% glycericacid, 48 hours reactivity=23% pyruvic acid and 47% glyceric acid. Upon48 h the yield of the reaction converting glyceric acid to pyruvic acidwas 23% (4.51 mM, 0.4 g/L). This reaction was scaled to a reactor withdimensions 14″ length×0.75″ outside diameter and 0.652″ inner diameter(Example 2.13 below). 20% conversion to product was observed (2 mMpyruvic acid) in 16 hours under the same conditions as described above

2.3 Production of acetyl phosphate from pyruvic acid using immobilizedpyruvate oxidase (PvOx) (FIG. 16): A reactor of 13.5″ in length with a0.25″ outside diameter and a 0.180″ internal diameter containingimmobilized PyOX enzyme (140 mg on 3.5 g of resin) was heated to 37° C.and allowed to equilibrate for 1 hour while passing equilibration buffer(10 mM Tris, 50 mM KH₂PO₄, 50 mM K₂HPO₄, pH 6.5, 5.0 mM MgCl2, and 100mM NaCl) through the reactor. After one hour had subsided, the substratesolution (10 mM Tris, 50 mM KH₂PO₄, 50 mM K₂HPO₄, pH 6.5, 5.0 mM MgCl₂,100 mM NaCl, 5 mM pyruvic acid, 5 mM thiamine pyrophosphate (TPP)) wasflowed through the reactor at a flow rate of 10 μL/min (72 min residencetime, FIG. 16). After the solution had passed through the reactor, itwas collected in the same beaker as the starting solution to allow thesolution to recycle through the individual reactor. The reaction wasallowed to proceed for 16 hours. The HPLC method was as follows: AnAgilent 1200 HPLC was equipped with a 30 cm Aminex HPX-87H column and amicro-guard cation H refill cartridge. The column was heated to 55° C.with the sample block being maintained at 25° C. The HPLC methodconsisted of 5 μl sample injection volume and an isocratic mobile phasecomprised of 100% sulfuric acid (10 mM). The run time was a total of 25minutes with acetyl phosphate eluting at 23.6 mins and pyruvate elutingat 16.0 minutes. A refractive index detector (Agilent) was used foranalysis after a two-hour equilibration period to produce a stablebaseline. Upon 16 h of reactivity, the reaction yield converting pyruvicacid into acetyl phosphate was 10% (0.5 mM or 92 mg/L). 00

2.4 Production of acetyl coenzyme A from acetyl phosphate usingimmobilized Acetyl-phosphate transferase (PTA) (FIG. 17): A reactor of13.5″ in length with a 0.25″ outside diameter and a 0.180″ internaldiameter containing immobilized PTA enzyme (112 mg on 3.50 g of resin)was heated to 55° C. and allowed to equilibrate for one hour whilepassing equilibration buffer (10 mM Tris, 50 mM KH₂PO₄, 50 mM K₂HPO₄, pH8.0, 5.0 mM MgCl₂, 100 mM NaCl) through the reactor. After 1 h hadsubsided, the substrate solution (10 mM Tris, 50 mM KH₂PO₄, 50 mMK₂HPO₄, pH 8.0, 5.0 mM MgCl₂, 100 mM NaCl, 3.2 mM acetyl phosphate, and3.2 mM CoA) was flowed through the reactor at a flow rate of 10 μL/min(72 min residence time, FIG. 17). After the solution had passed throughthe reactor, it was collected in the same beaker as the startingsolution to allow the solution to recycle through the individualreactor. The reaction was allowed to proceed for 8 h. For sampling, thereaction fluid was examined on a HPLC to examine the amount of acetylphosphate and acetyl-CoA. The HPLC method was as follows: An Agilent1200 HPLC was fitted with a HYPERSIL ODS COLUMN 150 mm×3 mm equippedwith a BetaSil C18 20 mm×2.1 mm guard column. The column was heated to25° C. with the sample block being maintained at 4° C. The HPLC methodused a 5 μl sample injection volume and a mobile phase comprised of 75mM CH₃COONa (sodium acetate) and 100 mM NaH₂PO4 (sodium dihydrogenphosphate) mixed with acetonitrile (94:6 volumetric ratio). The run timewas a total of 12 minutes with acetyl-coA eluting at 8.5 mins andcoenzyme A eluting at 3.9 minutes. A diode array detector (Agilent) wasused for the detection of the molecule of interest at 259 nm. Upon 8 hof reactivity, the reaction yield converting acetyl phosphate to acetylcoenzyme A was 12% (0.384 mM or 337 mg/L).

2.5 Production of acetoacetyl coenzyme A from acetyl co-enzyme A usingimmobilized Acetyl-CoA acetyltransferase (PhaA) (FIG. 18): A reactor of5.5″ in length with a 0.25″ outside diameter and a 0.218″ inner diametercontaining immobilized PhaA enzyme (41 mg on 1.5 g of resin) was heatedto 32° C. and allowed to equilibrate for 1 h while passing equilibrationbuffer (10 mM Tris, 50 mM KH₂PO₄, 50 mM K₂HPO₄, pH 8.0, 5.0 mM MgCl₂,100 mM NaCl) through the reactor. After 1 h had subsided, the substratesolution (10 mM Tris, 50 mM KH₂PO₄, 50 mM K₂HPO₄, pH 8.0, 5.0 mM MgCl₂,100 mM NaCl, 2.5 mM acetyl CoA) was flowed through the reactor at a flowrate of 10 μL/min (43 min residence time, FIG. 18). After the solutionhad passed through the reactor it was collected in the same beaker asthe starting solution to allow the solution to recycle through theindividual reactor. The reaction was allowed to proceed for 8 h. Forsampling, the reaction fluid was examined on the HPLC system to examinethe amount of AcCoA and CoA. The HPLC method was as follows: An Agilent1200 HPLC was fitted with a HYPERSIL ODS COLUMN 150 mm×3 mm equippedwith a BetaSil C18 20 mm×2.1 mm guard column. The column was heated to25° C. with the sample block being maintained at 4° C. HPLC methodconsisted of 5 μl sample injection volume and an isocratic gradientcomprised of 75 mM CH₃COONa and 100 mM NaH₂PO₄ mixed with acetonitrile(ACN) in a ratio 94:6 was used as the mobile phase. The run time was atotal of 12 minutes with acetyl-coA eluting at 8.5 mins and coenzyme Aeluting at 3.9 minutes. A diode array detector (Agilent) was used forthe detection of the molecule of interest at 259 nm. Upon 8 h ofreactivity, the reaction yield converting acetyl phosphate toacetoacetyl coenzyme A was 21% (0.94 mM or 930 mg/L).

2.6 Production of β-Hydroxy β-methylglutaryl-Coenzyme A (HMG-CoA) fromacetoacetyl coenzyme A using immobilized HMG-CoA Svnthase A110G (HMGSA110G) (FIG. 19): A reactor of 5.5″ in length with a 0.25″ outsidediameter and a 0.218″ inner diameter containing immobilized HMGS A110Genzyme (34.5 mg on 1.5 g of resin) was heated to 32° C. and allowed toequilibrate for 1 h while passing equilibration buffer (50 mM Tris, 100mM NaCl, 5 mM MgCl₂, pH 7.0) through the reactor. After 1 h hadsubsided, the substrate solution (50 mM Tris, 100 mM NaCl, 5 mM MgCl₂,pH 7.0, 5 mM acetoacetyl CoA) was flowed through the reactor at a flowrate of 10 μL/min (43 min residence time, FIG. 19). After the solutionhad passed through the reactor it was collected in the same beaker asthe starting solution to allow the solution to recycle through theindividual reactor. The reaction was allowed to proceed for 2 h. After 2h, the reaction solution was incubated with 20.7 μM HMGR and 5 mMnicotinamide adenine dinucleotide phosphate (NADPH). HMGR convertsacetoacetyl CoA into HMG-CoA using the cofactor NADPH. The activity ofHMGR was measured by monitoring loss of NADPH at 340 nm using aspectrophotometer. Upon 2 h of reactivity, the reaction yield convertingacetoacetyl CoA to HMG-CoA was 13% (0.65 mM or 621 mg/L).

2.7 Production of mevalonate from HMG-CoA using immobilized HMG-CoAReductase (HMGR) (FIG. 20): A reactor of 5.5″ in length with a 0.25″outside diameter and a 0.218″ inner diameter containing immobilized HMGSA110G enzyme (31 mg on 1.5 g of resin) was heated to 37° C. and allowedto equilibrate for 1 h while passing equilibration buffer (50 mM Tris,100 mM NaCl, 5 mM MgCl₂, pH 7.0) through the reactor. After 1 h hadsubsided, the substrate solution (50 mM Tris, 100 mM NaCl, 5 mM MgCl₂,pH 7.0, 5 mM NADPH) was flowed through the reactor at a flow rate of 10μL/min (43 min residence time, FIG. 20). After the solution had passedthrough the reactor it was collected in the same beaker as the startingsolution to allow the solution to recycle through the individualreactor. The activity of HMGR was measured by monitoring the loss ofNADPH at 340 nm using a spectrophotometer. Upon 2 h of reactivity, thereaction yield converting HMG-COA to mevalonate was 98.3% (2.45 mM or378 mg/L).

2.8 Production of Mevalonate-5-Phosphate from mevalonate usingimmobilized Melvonate Kinase (MVK) (FIG. 21): A reactor of 5.5″ inlength with a 0.25″ outside diameter and a 0.218″ inner diametercontaining immobilized MVK enzyme (60 mg on 1.5 g of resin) was heatedto 37° C. and allowed to equilibrate for 1 h while passing equilibrationbuffer (50 mM Tris, 5 mM MgCl₂, pH 8) through the reactor. After 1 h hadsubsided, the substrate solution (50 mM Tris, 5 mM MgCl₂, pH 8, 4 mMadenosine triphosphate (ATP), 4 mM mevalonic acid) was flowed throughthe reactor at a flow rate of 10 μL/min (43 min residence time, FIG.21). After the solution had passed through the reactor it was collectedin the same beaker as the starting solution to allow the solution torecycle through the individual reactor. The reaction was allowed toproceed for 16 hours. For sampling, the reaction fluid was examined on aHPLC system to examine the amount of ATP and ADP (adenosine diphosphate)present in the final reaction solution. The HPLC method was as follows:An Agilent 1200 HPLC was fitted with a HYPERSIL ODS COLUMN 150 mm×3 mmequipped with a BetaSil C18 20 mm×2.1 mm guard column. The column washeated to 25° C. with the sample block being maintained at 4° C. HPLCmethod consisted of 5 μl sample injection volume and an isocratic mobilephase comprised of 100 mM KH₂PO₄ (potassium dihydrogen phosphate), 8 mMTBAHS (tetrabutylammonium hydrogen sulfate), pH 6.0, 20% methanol (v/v).The run time was a total of 10 mins with ATP eluting at 5.7 mins and ADPeluting at 4.6 minutes. A diode array detector (Agilent) was used forthe detection of the molecule of interest at 254 nm. Upon 16 h ofreactivity, the reaction yields converting mevalonic acid to mevalonicacid-5-phosphate 0.3 mM (68 mg/L).

2.9 Production of Mevalonate-5-Disphosphate from Mevalonate-5-Phosphateusing Immobilized Phosphomevalonate Kinase (PMVK) (FIG. 22): A reactorof 5.5″ in length with a 0.25″ outside diameter and a 0.218″ innerdiameter containing immobilized PMVK enzyme (54 mg on 1.5 g of resin)was heated to 37° C. and allowed to equilibrate for 1 h while passingequilibration buffer (50 mM Tris, 5 mM MgCl₂, pH 8) through the reactor.After 1 h had subsided, the substrate solution (50 mM Tris, 5 mM MgCl₂,pH 8, 4 mM ATP, 4 mM mevalonic acid-5-phosphate) was flowed through thereactor at a flow rate of 10 μL/min (43 min residence time, FIG. 22).After the solution had passed through the reactor it was collected inthe same beaker as the starting solution to allow the solution torecycle through the individual reactor. The reaction was allowed toproceed for 32 hours. For sampling, the reaction fluid was examined on aHPLC system to examine the amount of ATP and ADP present in the reactionmixture. The HPLC method was as follows: An Agilent 1200 HPLC was fittedwith a HYPERSIL ODS COLUMN 150 mm×3 mm equipped with a BetaSil C18 20mm×2.1 mm guard column. The column was heated to 25° C. with the sampleblock being maintained at 4° C. HPLC method consisted of 5 μl sampleinjection volume and an isocratic mobile phase comprised of 100 mMKH₂PO₄, 8 mM TBAHS, pH 6.0, 20% methanol (v/v). The run time was a totalof 10 mins with ATP eluting at 5.7 mins and ADP eluting at 4.6 minutes.A diode array detector (Agilent) was used for the detection of themolecule of interest at 254 nm. Upon 32 h of reactivity, the reactionconverted mevalonic acid to mevalonic acid-5-pyrophosphate (2.1 mM, 697mg/L).

2.10 Production of Isopentenyl Pyrophosphate fromMevalonate-5-Diphosphate using Immobilized Diphosphomevalonate Kinase(MDC) (FIG. 23): A reactor of 5.5″ in length with a 0.25″ outsidediameter and a 0.218″ inner diameter containing immobilized MDC enzyme(30 mg on 1.5 g of resin) was heated to 37° C. and allowed toequilibrate for one hour while passing equilibration buffer (50 mM Tris,5 mM MgCl₂, pH 8.0) through the reactor. After 1 h had subsided, thesubstrate solution (50 mM Tris, 5 mM MgCl₂, pH 8, 4 mM ATP, 4 mMmevalonic acid-5-pyrophosphate) was flowed through the reactor at a flowrate of 10 μL/min (43 min residence time, FIG. 23). After the solutionhad passed through the reactor it was collected in the same beaker asthe starting solution to allow the solution to recycle through theindividual reactor. The reaction was allowed to proceed for 16 hours.For sampling, the reaction fluid was examined on the HPLC system toexamine the amount of ATP and ADP. The HPLC method was as follows: AnAgilent 1200 HPLC was fitted with a HYPERSIL ODS COLUMN 150 mm×3 mmequipped with a BetaSil C18 20 mm×2.1 mm guard column. The column washeated to 25° C. with the sample block being maintained at 4° C. HPLCmethod consisted of 5 μl sample injection volume and an isocraticgradient comprised of 100 mM KH₂PO₄, 8 mM TBAHS, pH 6.0, 20% methanol(v/v) was used as the mobile phase. The run time was a total of 10 minswith ATP eluting at 5.7 mins and ADP eluting at 4.6 minutes. A diodearray detector (Agilent) was used for the detection of the molecule ofinterest at 254 nm. Upon 16 h of reactivity, mevalonicacid-5-pyrophosphate was converted into isopentenyl pyrophosphate at 0.9mM (237 mg/L).

2.11 Production of Geranyl Pyrophosphate (GPP) from Dimethylallylpyrophosphate using immobilized Farnesvl-PP svnthase (FPPS) and areporter prenyl transferase for CBGA production (FIG. 24): A reactor of5.5″ in length with a 0.25″ outside diameter and a 0.218″ inner diametercontaining immobilized FPPS enzyme (68 mg on 1.5 g of resin) was heatedto 25° C. and allowed to equilibrate for 1 h while passing equilibrationbuffer (50 mM Tris pH 8.0, 5 mM MgCl₂, 10 mM NaCl) through the reactor.After 1 h had subsided, the substrate solution (50 mM Tris, pH 8, 5 mMMgCl₂, 10 mM NaCl, 3.5 mM isopentenyl pyrophosphate, 3.5 mMdimethylallyl pyrophosphate) was flowed through the reactor at a flowrate of 10 μL/min with an intermittent pausing of 50 sec after each 10seconds of pumping to meet the residence time of 4 hours (FIG. 24).Unlike other examples above, the reaction solution was not recycled inthe experiment. The reaction solution collected after the completion ofdesignated time was incubated with 3.5 mM olivetolic acid (OA) and 120μM prenyl transferase (NphB) for 2 hours at 25° C. Completed reactionswere extracted 3× with ethyl acetate, evaporated, and resuspended inmethanol for analysis on a HPLC system to examine the amount of CBGA(cannabigerolic acid). The coupled reaction was able to convert 25% ofthe starting material to the product. The HPLC method was as follows: AnAgilent 1200 HPLC was fitted with a 250 mm×4.6 mm, 5 μm liChrospher RP8column equipped with a guard column. The column was heated to 30° C.with the sample block being maintained at 25° C. HPLC method consistedof 5 μl sample injection volume and an isocratic mobile phase comprisedof 25% buffer A (water, 0.1% formic acid, 5 mM ammonia formate) and 75%buffer B (acetonitrile, 0.1% formic acid, 5 mM ammonia formate) was usedas the mobile phase. CBGA produced in the reaction was measured usingDAD at 228 nm. The run time was a total of 10 minutes with CBGA elutingat 3.68 mins. Upon the analysis of the coupled reactions, the reactionyielded 0.9 mM or 285 mg/L of the final product, CBGA.

2.12 Removal of oxygen from a reaction stream prior to an individualreactor: In some instances, the partial pressure of gaseous oxygen inthe reaction mixture must be decreased to avoid enzyme deactivation.Thus, prior to a reaction solution being pumped through a reactor, thesolution must be pumped through a deoxygenation and/or degassing deviceto decrease the partial pressure of oxygen in the reaction solutionprior to the next reaction. The use of a deoxygenation and/or degassingmachine when coupled with the system allowed the removal of up to 3.68ppm of oxygen, a reduction of almost 50% (FIG. 17). For testing,deionized water passed through the deoxygenation/degassing device at aflow rate of 0.25 mL/min. This process takes approximately 20 mins tocomplete. In addition to the deoxygenation/degassing component, nitrogengas may be introduced into a solution to further remove oxygen. Nitrogengas input controlled by a mass flow controller is added to a solutionprior to being pumped through a reactor (FIGS. 25A-25B).

2.13 Multi-step enzyme reaction to convert glycerol to pyruvic acid(FIG. 26): A reactor of 14″ in length with a 0.75″ outside diameter anda 0.652″ internal diameter containing immobilized Aldo enzyme (2.7 genzyme on 56 g of resin) was heated to 37° C. and equilibrated for sixhours with equilibration buffer (50 mM tris, pH 8.5) by pumping itthrough the reactor. After this time, the substrate solution (40 mL, 50mM Tris, pH 8.5 20 mM glycerol) was flown through the reactor at a flowrate of 10 μL/min. Prior to entering the reactor, the substrate solutionwas mixed with an equal flow rate of compressed air (0.01 sccm) to yielda total flow rate through the reactor of 20 μL/min (8 hour residencetime). After the solution has passed through the reactor, the fluid flow(now containing only glyceric acid, 98% conversion from the previousreactor) is pH adjusted in-line. For this, pH buffer (50 mM Tris, pH12.0) was added into the reaction flow using a T-piece mixer with a pHbuffer solution flow rate of 10 μL/min. The resulting fluid now pHadjusted is held momentarily in a stirred tank reactor (CSTR) where itwas degassed by using a bubbling nitrogen flow. Finally, another pumpdraws the fluid from the CSTR into the second reactor of 14″ in lengthwith a 0.75″ outside diameter and a 0.652″ internal diameter containingimmobilized DHAD enzyme (1.8 g enzyme on 56 g of resin) heated at 45° C.The total flow rate of reaction mixture through the reactor was 10μL/min (16.25-hour residence time). The solution was collected at theend of the second reactor into a glass beaker. The reaction mixture wasthen analyzed every 24 hours. For sampling, 100 μl of the reaction fluidwas examined on a HPLC system to examine the amount of glycerol,glyceric acid, and pyruvic acid. The HPLC method was as follows: AnAgilent 1200 HPLC was equipped with a 30 cm Aminex HPX-87H column and amicro-guard cation H refill cartridge. The column was heated to 55° C.and the sample block was maintained at 25° C. For each sample, 1 μL wasinjected and an isocratic mobile phase comprised of 100% sulfuric acid(10 mM) was used. The sample run time was a total of 45 minutes withglyceric acid eluting at 17.2 mins, glycerol eluting at 21.0 minutes,and pyruvic acid eluting at 15.8 mins. For detection, a RID detector(Agilent) was used after a 2 h equilibration period produced a stablebaseline. It was found that the first reactor converted 98% of theglycerol to glyceric acid and the second reactor converted the glycericacid to pyruvic acid at a final concentration of 2 mM. We found that theALDO reactor was able to achieve 98% conversion levels for 3 days withno loss in productivity and remained able to do the enzyme conversionfor 10 days of continual processing.

EMBODIMENTS

The following embodiments are intended to be illustrative only and notto be limiting in any way.

Embodiment 1: A cell-free manufacturing platform for chemicalproduction, the platform comprising: one or more individual reactors,wherein each of the one or more individual reactors comprises: acylindrical tube comprising a first end and a second end, wherein boththe first end of the cylindrical tube and the second end of thecylindrical tube comprise fittings, wherein a cylindrical tube interiorof the individual reactor comprises: a resin and an enzyme and, apumping system adapted to flow a solution through the one or moreindividual reactors, wherein each of the one or more individual reactorshas an input tubing connected at the first end of the cylindrical tubeand an output tubing connected at the second end of the cylindrical tubeto create a closed system.

Embodiment 2: The platform of Embodiment 1, wherein the fittings arestainless steel fittings.

Embodiment 3: The platform of Embodiment 1, wherein the cylindrical tubeinterior of the individual reactor further comprises one or moresensors.

Embodiment 4: The platform of Embodiment 3, wherein the one or moresensors comprises a temperature sensor, a pH sensor, a pressure sensor,a flow rate sensor, a dissolved oxygen (DO) sensor, a spectrometer, or acombination thereof.

Embodiment 5: The platform of any one of Embodiments 1-4, wherein eachof the one or more individual reactors comprises an individual reactorhousing.

Embodiment 6: The platform of Embodiment 5, wherein the individualreactor housing surrounds and is fastened to the individual reactor.

Embodiment 7: The platform of any one of Embodiments 1-6, furthercomprising a temperature altering element attached to the individualreactor housing or the individual reactor.

Embodiment 8: The platform of Embodiment 7, wherein the temperaturealtering element is a thermoelectric cooler (TEC).

Embodiment 9: The platform of any one of Embodiments 1-8, furthercomprising a spectrometer attached in series with the one or moreindividual reactors.

Embodiment 10: The platform of any one of Embodiments 1-9, furthercomprising a degassing module adapted to remove gasses from thesolution.

Embodiment 11: The platform of Embodiment 10, wherein the degassingmodule is a deoxygenation module.

Embodiment 12: The platform of Embodiment 11, wherein the deoxygenationmodule is adapted to remove oxygen from the solution.

Embodiment 13: The platform of any one of Embodiments 1-12, furthercomprising a gas addition module adapted to introduce gas into thesolution.

Embodiment 14: The platform of any one of Embodiments 1-13, furthercomprising a pH module adapted to introduce an acid or base into thesolution

Embodiment 15: The platform of any one of Embodiments 1-14, furthercomprising a graphical user interface (GUI) adapted to controlautomation software and hardware

Embodiment 16: The platform of any one of Embodiments 1-15, wherein thecell-free manufacturing platform is able to automatically change each ofthe one or more reactors conditions based on input from the sensors.

Embodiment 17: The platform of any one of Embodiments 1-16, wherein theenzyme comprises: MBP-Aldo (Aldo), Dihydroxy Acid Dehydratase (TvDHAD),Pyruvate Oxidase (PyOx), Acetyl-phosphate transferase (PTA), Acetyl-CoAacetyltransferase (PhaA), HMG-CoA Synthase A110G (HMGS), HMG-CoAReductase (HMGR), Mevalonate Kinase (MVK), Phosphomevalonate Kinase(PMVK), Diphosphomevalonate Kinase (MDC), Isopentyl-PP Isomerase (IDI),Farnesyl-PP synthase S82F (FPPS), Prenyl transferase (NphB) or acombination thereof.

Embodiment 18: The platform of any one of Embodiments 1-17, wherein theenzymes are immobilized.

Embodiment 19: The platform of any one of Embodiments 1-17, wherein theenzymes are non-immobilized

As used herein, the term “about” refers to plus or minus 10% of thereferenced number.

Although there has been shown and described the preferred embodiment ofthe present invention, it will be readily apparent to those skilled inthe art that modifications may be made thereto which do not exceed thescope of the appended claims. Therefore, the scope of the invention isonly to be limited by the following claims. In some embodiments, thefigures presented in this patent application are drawn to scale,including the angles, ratios of dimensions, etc. In some embodiments,the figures are representative only and the claims are not limited bythe dimensions of the figures. In some embodiments, descriptions of theinventions described herein using the phrase “comprising” includesembodiments that could be described as “consisting essentially of” or“consisting of”, and as such the written description requirement forclaiming one or more embodiments of the present invention using thephrase “consisting essentially of” or “consisting of” is met.

What is claimed is:
 1. A cell-free manufacturing platform for chemicalproduction, the platform comprising: a) one or more individual reactors,wherein each of the one or more individual reactors comprises: acylindrical tube comprising a first end and a second end, wherein boththe first end of the cylindrical tube and the second end of thecylindrical tube comprise fittings, wherein an input tubing is connectedat the first end of the cylindrical tube and an output tubing isconnected at the second end of the cylindrical tube, wherein acylindrical tube interior of the individual reactor comprises: i) aresin; and ii) an enzyme; and b) a pumping system adapted to flow asolution through the one or more individual reactors.
 2. The platform ofclaim 1, wherein the fittings are stainless steel fittings.
 3. Theplatform of claim 1, wherein the cylindrical tube interior of theindividual reactor further comprises one or more sensors.
 4. Theplatform of claim 3, wherein the one or more sensors comprises atemperature sensor, a pH sensor, a pressure sensor, a flow rate sensor,a dissolved oxygen (DO) sensor, a spectrometer, or a combinationthereof.
 5. The platform of claim 1, wherein each of the one or moreindividual reactors comprises an individual reactor housing.
 6. Theplatform of claim 5, wherein the individual reactor housing surroundsand is fastened to the individual reactor.
 7. The platform of claim 1,further comprising a temperature altering element attached to theindividual reactor housing or the individual reactor.
 8. The platform ofclaim 7, wherein the temperature altering element is a thermoelectriccooler (TEC).
 9. The platform of claim 1, further comprising aspectrometer attached in series with the one or more individualreactors.
 10. The platform of claim 1, further comprising a degassingmodule adapted to remove gasses from the solution.
 11. The platform ofclaim 10, wherein the degassing module is a deoxygenation module. 12.The platform of claim 11, wherein the deoxygenation module is adapted toremove oxygen from the solution.
 13. The platform of claim 1, furthercomprising a gas addition module adapted to introduce gas into thesolution.
 14. The platform of claim 1, further comprising a pH moduleadapted to introduce an acid or base into the solution.
 15. The platformof claim 1, further comprising a graphical user interface (GUI) adaptedto control automation software and hardware.
 16. The platform of claim1, wherein the cell-free manufacturing platform is able to automaticallychange each of the one or more reactors conditions based on input fromthe sensors.
 17. The platform of claim 1, wherein the enzyme comprises:MBP-Aldo (Aldo), Dihydroxy Acid Dehydratase (TvDHAD), Pyruvate Oxidase(PyOx), Acetyl-phosphate transferase (PTA), Acetyl-CoA acetyltransferase(PhaA), HMG-CoA Synthase A110G (HMGS), HMG-CoA Reductase (HMGR),Mevalonate Kinase (MVK), Phosphomevalonate Kinase (PMVK),Diphosphomevalonate Kinase (MDC), Isopentyl-PP Isomerase (IDI),Farnesyl-PP synthase S82F (FPPS), Prenyl transferase (NphB) or acombination thereof.
 18. The platform of claim 1, wherein the enzymesare immobilized.
 19. The platform of claim 1, wherein the enzymes arenon-immobilized.